Site-directed mutagenesis of the endo-beta-1,4-glucanase (EG) gene from the termite Reticulitermes speratus EG (RsEG) was performed to gain a better understanding of the role of certain amino acid residues in the activity of the enzyme. Three mutants, G91A, Y97W and K429A had higher activities towards carboxymethyl cellulose than the wild type. The mutations had synergistic effects since each single mutant exhibited about 3-4-fold of wild type activity, but the corresponding activities for double and triple mutants were 7-13-fold. Mutant G147R lost the enzymatic activity completely, suggesting G147 plays a significant role in maintaining enzyme activity. The predicted roles of Asp53, Asp56 and Glu411 in enzymatic catalysis were experimentally verified since the resultant mutants lost the enzyme activities. This study presents the first report on the relationship between amino acid residues and enzyme activity of termite EG, and the information will potentially be useful for industrial application of termite-origin cellulase. (C) 2010 Elsevier Ltd. All rights reserved.